Multiblock biodegradable hydrogels for drug delivery and tissue treatment

ABSTRACT

Gel-forming macromers including at least four polymeric blocks, at least two of which are hydrophobic and at least one of which is hydrophilic, and including a crosslinkable group are provided. The macromers can be covalently crosslinked to form a gel on a tissue surface in vivo. The gels formed from the macromers have a combination of properties including thermosensitivity and lipophilicity, and are useful in a variety of medical applications including drug delivery and tissue coating.

This application is a continuation-in-part of U.S. Ser. No. 60/001,723,filed Jul. 28, 1995.

BACKGROUND OF THE INVENTION

The present invention is generally in the area of biodegradable polymersfor use in drug delivery and biomedical applications.

Biodegradable polymers have been developed for use in a variety ofsurgical and drug delivery applications. The synthesis andbiodegradability of poly(lactic acid) was reported by Kulkarni et al.,Arch. Surg., 93:839 (1966). Biodegradable polyanhydrides andpolyorthoesters having labile backbone linkages have been developed.Domb et al., Macromolecules, 22:3200 (1989); and Heller et al.,“Biodegradable Polymers as Drug Delivery Systems,” Chasin, M. andLanger, R., Eds., Dekker, N.Y., 121-161 (1990), the disclosures of whichare incorporated herein. Polymers which degrade into naturally occurringmaterials, such as polyaminoacids, also have been developed. Polyestersof α-hydroxy acids, such as lactic acid or glycolic acid, are widelyused as biodegradable materials for applications ranging from closuredevices, including sutures and staples, to drug delivery systems.Holland et al., Controlled Release, 4:155-180, (1986); U.S. Pat. No.4,741,337 to Smith et al.; and Spilizewski et al., J. Control. Rel.,2:197-203 (1985), the disclosures of which are incorporated herein.

Degradable polymers containing water-soluble polymer elements have beendescribed. Degradable polymers have been formed by copolymerization oflactide, glycolide, and ε-caprolactone with the polyether, polyethyleneglycol (“PEG”), to increase the hydrophilicity and degradation rate.Sawhney et al., J. Biomed. Mater. Res. 24:1397-1411 (1990). U.S. Pat.No. 4,716,203 to Casey et al. describes the synthesis of a blockcopolymer of PGA (poly(glycolic acid)) and PEG. U.S. Pat. No. 4,716,203to Casey et al. describes the synthesis of PGA-PEG diblock copolymers.

Polymers formed from crosslinkable monomers or prepolymers have beendeveloped in the prior art. Crosslinked hyaluronic acid has been used asa degradable swelling polymer for biomedical applications. U.S. Pat.Nos. 4,987,744 and 4,957,744 to Della Valle et al.; and Della Valle etal., Polym. Mater. Sci. Eng., 62:731-735 (1991).

U.S. Pat. No. 5,410,016 to Hubbell et al., the disclosure of which inincorporated herein, discloses the in situ crosslinking ofbiodegradable, water-soluble macro-monomers, (“macromers”) to formbarrier coatings and matrices for delivery of biologically activeagents. Other polymers for drug delivery or other biomedicalapplications are described in U.S. Pat. No. 4,938,763 to Dunn, U.S. Pat.Nos. 5,160,745 and 4,818,542 to DeLuca, U.S. Pat. No. 5,219,564 toZalipsky, U.S. Pat. No. 4,826,945 to Cohn, and U.S. Pat. Nos. 5,078,994and 5,429,826 to Nair, the disclosures of which are incorporated hereinby reference. Methods for delivery of the polymers materials includesyringes (U.S. Pat. No. 4,938,763 to Dunn et al.) spray applicators (WO94/21324 by Rowe et al.) and catheter delivery systems (U.S. Pat. Nos.5,328,471; and 5,213,580 to Slepian). The synthesis of macromersincluding a central chain of polyethylene glycol, with an oligomerichydroxyacid at each end and acrylic esters at the ends of the hydroxyacid oligomer also has been reported. Sawhney A. S. et al.,Macromolecules, 26: 581 (1993); and PCT WO 93/17669 by Hubbell J. A. etal., the disclosures of which are incorporated herein by reference.

Thermal volume changes in polymeric gels, such as esters and amides ofpolyacrylic acid, have been described. For example, poly(N-isopropylacrylamide) based hydrogels, which are thermosensitive in aqueoussystems, have been used for controlled drug delivery and otherapplications. U.S. Pat. No. 5,403,893 to Tanaka et al.; and Hoffman A.S. et al., J. Controlled Release, 6:297 (1987), the disclosures of whichare incorporated herein. Poly(N-isopropyl acrylamide), however, isnon-degradable and is not suitable for applications where biodegradablepolymers are required. Non-biodegradable polymeric systems for drugdelivery are disadvantageous since they require removal after thedrug-polymer device is implanted.

It is an object of the invention to provide improved polymer systems foruse in drug delivery and other biomedical applications such as surgicalapplications. It is an additional object of the invention to providepolymer systems for use in controlled drug delivery which are capable ofreleasing a biologically active agent in a predictable and controlledrate. It is a further object of the invention to provide polymers foruse in controlled drug delivery which release the active agent locallyat a particular targeted site where it is needed. It is another objectof the invention to provide polymer systems for use in drug deliverywhich have properties including volume and drug release which arevariable with temperature or other parameters such as pH or ionconcentration.

SUMMARY OF THE INVENTION

Macromers are provided which are capable of gelling in an aqueoussolution. In one embodiment, the macromers include at least fourpolymeric blocks, at least one of which is hydrophilic and at least twoof which are hydrophobic, and include a crosslinkable group. The polymerblocks may be selected to provide macromers with different selectedproperties. The macromers can be covalently crosslinked to form a gel ona tissue surface in vivo. The gels formed from the macromers have acombination of properties including thermosensitivity and lipophilicity,and are useful in a variety of medical applications including drugdelivery and tissue coating.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a scheme showing the different gel states and properties ofone embodiment of a thermoresponsive biodegradable macromer formed froma polypropylene oxide-polyethylene oxide block copolymer.

FIG. 2 is a graph of temperature-dependent changes in gel volume of gelsformed by photopolymerization of an acrylated polypropyleneoxide-polyethylene oxide block copolymer containing a biodegradableregion.

FIG. 3 is a graph showing the effects of temperature on dextran releasefrom a gel formed by photopolymerization of an acrylated polypropyleneoxide-polyethylene oxide block copolymer.

FIG. 4 is a graph illustrating the variation in the speed ofphotocrosslinking of acrylated polypropylene oxide-polyethylene oxideblock copolymers having incorporated therein different biodegradableregions.

FIG. 5 is a graph showing the in vitro profiles of degradation rate ofgels formed by photocrosslinking of acrylated polypropyleneoxide-polyethylene oxide block copolymers having incorporated thereindifferent biodegradable regions.

FIG. 6 is a graph illustrating the biocompatibility of gels formed byphotocrosslinking acrylated polypropylene oxide-polyethylene oxide blockcopolymers having incorporated therein different biodegradable regions.

FIG. 7 shows graphs illustrating release of fluorescent dextran fromgels formed by photocrosslinking acrylated polypropyleneoxide-polyethylene oxide block copolymers having incorporated thereinbiodegradable linkers.

FIG. 8 shows graphs of transition temperatures of gels formed frommacromers containing biodegradable linkers.

FIG. 9 illustrates the chemical structures of biodegradablecrosslinkable macromers consisting of acrylated poly(propyleneoxide)-poly(ethylene oxide) block copolymers having incorporated thereina biodegradable linker.

FIG. 10 is a graph of absorbance of a hydrophobic dye vs. log (weight %)of solutions of biodegradable macromers having a hydrophobic regionincorporated therein.

FIG. 11 is a schematic illustration of a cell membrane includinghydrophobic bilayer with a macromer including a hydrophobic taildiffused into the bilayer.

FIG. 12 is a schematic illustration of nanospheres or microspheres whichcan be formed by aggregation and subsequent polymerization ofhydrophilic macromers.

FIG. 13 is a graph which shows the rate of release of a small drug fromgels formed from hydrophobic macromers.

FIGS. 14 and 15 are graphs showing diffusivity of a sparingly watersoluble drug through a hydrophobic hydrogel.

FIG. 16 is a graph showing the release of tetracycline from a hydrogelformed from monomers including a biodegradable region.

DETAILED DESCRIPTION OF THE INVENTION

Macromers are provided which are crosslinkable to form hydrogels whichare useful as matrices for controlled drug delivery. In a preferredembodiment, biodegradable macromers are provided in a pharmaceuticallyacceptable carrier, and are capable of crosslinking, covalently ornon-covalently, to form hydrogels which are thermoresponsive. Abiologically active agent may be incorporated within the macromersolution or in the resulting hydrogel after crosslinking. The hydrogelshave properties, such as volume and drug release rate, which aredependent upon temperature. The hydrogels may be formed in situ, forexample, at a tissue site, and may be used for for controlled deliveryof bioactive substances and as tissue coatings. The macromers used toform the hydrogels may be fabricated with domains having specificproperties including selected hydrophobicity, hydrophilicity,thermosensitivity or biodegradability, and combinations thereof.

Macromers

The macro-monomers (“macromers”) which are ionically or covalentlycrosslinkable to form hydrogels preferably consist of a block copolymer.The macromers can be quickly polymerized from aqueous solutions. Themacromers are advantageously capable of thermoreversible gelationbehavior, and preferably may be polymerized in a solution state or in agel state. The macromers are defined as including a hydrophilic blockcapable of absorbing water, and at least one block, distinct from thehydrophilic block, which is sufficiently hydrophobic to precipitatefrom, or otherwise change phase while within, an aqueous solution,consisting of water, preferably containing salts, buffers, drugs orpolymerizing reagents, at temperatures within or near thephysiologically compatible range, for example 0 to 65° C. Thehydrophilic block optionally may be an amphiphilic block. The macromermay include more than one of the same or different hydrophilic orhydrophobic region. Preferably, the macromers include at least threeblocks, or more preferably four blocks.

The block copolymers may be linear (AB, ABA, ABABA or ABCBA type), star(AnB or BAnC, where B is at least n-valent, and n is 3 to 6) or branched(multiple A's depending from one B). In these formulae, either A or Bmay be the hydrophilic block, and the other the amphipathic orhydrophilic block, and the additional block C may be either.

In another embodiment, the macromer includes at least fourcovalently-linked polymeric blocks, wherein: at least one, or in anotherembodiment, at least two blocks are hydrophilic, and the hydrophilicblocks individually have a water solubility of at least 1 gram/liter; atleast two blocks are sufficiently hydrophobic to aggregate to formmicelles in an aqueous continuous phase; and the macromer furtherincludes at least one crosslinkable group. The crosslinkable groupsoptionally may be separated by at least one degradable linkage capableof degrading under physiological conditions. In one embodiment, at leastone hydrophobic block may be separated from any reactive group by atleast one hydrophilic block.

The macromer further may include five total blocks having the same ordifferent properties such as thermal sensitivity, hydrophilicity orhydrophobicity. Each block also may have a combination of properties.For example, a block may be hydrophilic and also thermosensitive.Additionally, the multiblock macromer may include chemically distinctblocks or may incorporate more than one of the same identical block. Themacromer is fabricated with a structure and with properties suitable fordifferent applications. For example the macromer may include a centralblock of dimer fatty acid which includes central hydrocarbon chain ofabout 30 carbon atoms and two terminal carboxy groups which areesterified with a thermosensitive poloxamer, such as Pluronic L1050 Thiscentral molecule further is polylactated at each hydroxy terminus, andend capped with acryloyl chloride. An another embodiment is a poloxamerincluding polyhydroxy groups polymerized on each end, and wherein themolecule is end capped at each end with a reactive group such as anacrylate or a secondary isocyanate.

The configuration of the macromers may be preselected depending on theuse of the macromer. The macromers may include at least two hydrophobicblocks, separated by a hydrophilic block. The macromers also may befabricated with a crosslinkable group which is separated by a degradablegroup from any other crosslinkable group. One preferred embodiment iswherein the dry macromer absorbs at least about 10% in weight of water.The molecular weight of the macromer preferably is at least 1000Daltons, or optionally is at least 2000 Daltons, or in an alternativeembodiment, at least 4000 Daltons.

In a preferred embodiment, the macromer includes at least one thermallysensitive region, and an aqueous solution of the macromer is capable ofgelling either ionically and/or by covalent crosslinking to produce ahydrogel with a temperature dependent volume. This permits the rate ofrelease of a drug incorporated in the hydrogel to change depending uponthe volume of the hydrogel. Useful macromers are those which are, forexample, capable of thermoreversible gelation of an aqueous solution ofthe macromer at a concentration of at least 2% by weight, and whereinthe gelation temperature is between about 0° C. and about 65° C. Themacromer also may have a phase transition temperature in the range of 0to 100° C., and wherein the transition temperature is affected by theionic composition of an aqueous solution of the macromer or theconcentration of macromer in the aqueous solution.

The macromers may be formed by modification of materials and methodsdescribed in the prior art Macromers including a central chain ofpolyethylene glycol, with oligomeric hydroxy acid at each end andacrylic esters at the ends of the hydroxy acid oligomer are described inSawhney A. S. et al., Macromolecules, 26: 581 (1993); and PCT WO93/17669 by Hubbell J. A. et al., the disclosures of which areincorporated herein by reference. U.S. Pat. No. 5,410,016 to Hubbell etal., the disclosure of which is incorporated herein by reference,discloses that biodegradable, water-soluble macromers can be crosslinkedin situ to form barrier coatings and depots or matrices for delivery ofbiologically active agents such as therapeutic drugs. In addition to thematerials and methods described in U.S. Pat. No. 5,410,016, materialsand methods described by Dunn (U.S. Pat. No. 4,938,763), DeLuca (U.S.Pat. Nos. 5,160,745; and 4,818,542), Zalipsky (U.S. Pat. No. 5,219,564),Cohn (U.S. Pat. No. 4,826,945), Nair (U.S. Pat. Nos. 5,078,994; and5,429,826), the disclosures of which are incorporated herein byreference, are useful to form the macromers described herein.

For example, the macromer may include a poloxamer backbone extended withhydrophobic materials, such as oligolactate moieties, which serve as thebiodegradable segment of the molecule, wherein the PEO-PPO-PEO-lactatecopolymer is terminated by acrylate moieties. The materials can becombined with, then delivered and photopolymerized in situ, onto targetorgans to conform to a specific shape.

The macromers and hydrogels formed therefrom preferably arebiocompatible, preferably not causing or enhancing a biological reactionwhen implanted or otherwise administered within a mammal. The macromers,and any breakdown products of the hydrogels or macromers, preferably arenot significantly toxic to living cells, or to organisms. The hydrogelsalso may have liquid crystalline properties for example at highconcentration, which are useful in controlling the rate of drugdelivery. Ionic properties can be provided in the backbone of themacromers, conferring the further property of control of delivery and/orphysical state by control of the ionic environment, including pH, of themacromer or gel. In one embodiment, the critical ion composition is thehydrogen ion concentration. For example, when a poloxamine, such as aTetronic surfactant, is used as the core of the macromer, then theresulting macromer has the ionic groups (amines) in the core, and themacromers' ability to gel upon changes in temperature is affected by thepH of the solution.

Thermosensitive Regions

The macromers may be provided with one or more regions which haveproperties which are thermoresponsive. As used herein,thermoresponsiveness is defined as including properties of a hydrogel,such as volume, transition from a liquid to a gel, and permeability tobiologically active agents, which are dependent upon the temperature ofthe hydrogel. In one embodiment, the macromers are capable of reversiblegelation which is controlled by temperature. The reversible gel furtheroptionally may be crosslinked in situ into an irreversibly andcovalently crosslinked gel. This permits the macromer to be appliedreliably in surgical applications on a specific area of tissue withoutrunning off or being washed off by body fluids prior to gelation orcrosslinking.

In one preferred embodiment, the macromers are capable of gellingthermoreversibly, for example, due to the content of poloxamer regions.Since gelling is thermoreversible, the gel will dissipate on cooling.The macromers may further include crosslinkable groups which permit thegel to be further covalently crosslinked for example byphotopolymerization. After crosslinking, the gels are irreversiblycrosslinked. However, they retain other significant thermoresponsiveproperties, such as changes in volume and in permeability.

By appropriate choice of macromer composition, hydrogels can be createdin situ which have thermosensitive properties, including volume changesand drug release which are dependent upon temperature, which can be usedto control drug delivery from the hydrogel. Control of drug delivery canbe further controlled by adjustment of properties such as hydrophobicityof amphiphilic or other regions in the gel. Change in volume of thehydrogel may readily be measured by examination of macroscopicunrestrained samples during temperature excursions. Changes in excess of100% in volume may be obtained with hydrogels formed from the macromers,such as an acrylate-capped polyglycolide-derivatized poloxamer of about30% PPO (polypropylene oxide) content, the expansion occurring graduallyon change of the temperature from about 0° C. to body temperature (37°C.). Changes of more than 5% in any linear dimension may be effective inaltering the release rate of a macromolecular drug.

The macromers preferably include thermogelling macromers, such as“poloxamers”, i.e., poly(ethylene oxide)-poly(propyleneoxide)-poly(ethylene oxide)(“PEO-PPO-PEO”), block copolymers. Aqueouspolymeric solutions of poloxamers undergo microphase transitions at anupper critical solution temperature, causing a characteristic gelformation. This transition is dependent on concentration and compositionof the block copolymer. Alexandridis et al., Macromolecules, 27:2414(1994). The segmental polyether portion of the molecule gives watersolubility and thermosensitivity. The material also advantageously havebeen demonstrated to be biocompatible.

For example, the macromer may include a poloxamer backbone extended withhydrophobic materials, such as oligolactate moieties, which serve as thebiodegradable segment of the molecule, wherein the PEO-PPO-PEO-lactatecopolymer is terminated by acrylate moieties. The materials can becombined with a bioactive agent, then delivered and photopolymerized insitu. In addition to poloxamer cores, meroxapols, such as “reversedPluronics” (PPO-PEO-PPO copolymers) and poloxamines, such as Tetronic™surfactants, may be used.

Other polymer blocks which may be provided in the monomer which arecapable of temperature dependent volume changes include water solubleblocks such as polyvinyl alcohol, polyvinyl-pyrrolidone, polyacrylicacids, esters and amides, soluble celluloses, peptides and proteins,dextrans and other polysaccharides. Additionally, polymer blocks with anupper critical point may be used, such as other polyalkylene oxides,such as mixed polyalkylene oxides and esters, derivatized celluloses,such as hydroxypropylmethyl cellulose, and natural gums such as konjacglucomannan.

In another embodiment, the macromer is defined as having an opticallyanisotropic phase at a concentration at or below the maximal solubilityof the macromer in an aqueous solution, at a temperature between about 0and 65° C.

Crosslinkable Groups.

The macromers preferably include crosslinkable groups which are capableof forming covalent bonds with other compounds while in aqueoussolution, which permit crosslinking of the macromers to form a gel,either after, or independently from thermally dependent gellation of themacromer. Chemically or ionically crosslinkable groups known in the artmay be provided in the macromers. The crosslinkable groups in onepreferred embodiment are polymerizable by photoinitiation by freeradical generation, most preferably in the visible or long wavelengthultraviolet radiation. The preferred crosslinkable groups areunsaturated groups including vinyl groups, allyl groups, cinnamates,acrylates, diacrylates, oligoacrylates, methacrylates, dimethacrylates,oligomethoacrylates, or other biologically acceptable photopolymerizablegroups.

Other polymerization chemistries which may be used include, for example,reaction of amines or alcohols with isocyanate or isothiocyanate, or ofamines or thiols with aldehydes, epoxides, oxiranes, or cyclic imines;where either the amine or thiol, or the other reactant, or both, may becovalently attached to a macromer. Mixtures of covalent polymerizationsystems are also contemplated. Sulfonic acid or carboxylic acid groupsmay be used.

Preferably, at least a portion of the macromers will have more than onecrosslinkable reactive group, to permit formation of a coherent hydrogelafter crosslinking of the macromers. Up to 100% of the macromers mayhave more than one reactive group. Typically, in a synthesis, thepercentage will be on the order of 50 to 90%, for example, 75 to 80%.The percentage may be reduced by addition of small co-monomerscontaining only one active group. A lower limit for crosslinkerconcentration will depend on the properties of the particular macromerand the total macromer concentration, but will be at least about 3% ofthe total molar concentration of reactive groups. More preferably, thecrosslinker concentration will be at least 10%, with higherconcentrations, such as 50% to 90%, being optimal for maximumretardation of many drugs. Optionally, at least part of the crosslinkingfunction may be provided by a low-molecular weight crosslinker. When thedrug to be delivered is a macromolecule, higher ranges of polyvalentmacromers (i.e., having more than one reactive group) are preferred. Ifthe gel is to be biodegradable, as is preferred in most applications,then the crosslinking reactive groups should be separated from eachother by biodegradable links. Any linkage known to be biodegradableunder in vivo conditions may be suitable, such as a degradable polymerblock. The use of ethylenically unsaturated groups, crosslinked by freeradical polymerization with chemical and/or photoactive initiators, ispreferred as the crosslinkable group.

The macromer may also include an ionically charged moiety covalentlyattached to the macromer, which optionally permits gellation orcrosslinking of the macromer.

Hydrophobic Regions

The macromers further may include hydrophobic domains. Thehydrophobicity of the gel may be modified to alter drug delivery orthree dimensional configuration of the gel. Amphiphilic regions may beprovided in the macromers which in aqueous solution tend to aggregate toform micellar domain, with the hydrophobic regions oriented in theinterior of these domains (the “core”), while the hydrophilic domainsorient on the exterior (“the corona”). These microscopic “cores” canentrap hydrophobic drugs, thus providing microreservoirs for sustaineddrug release. Kataoka K., et al., J. Controlled Release, 24:119 (1993).The fundamental parameter of this supramolecular assemblage ofamphiphilic polymers in aqueous solution is the Critical MicellarConcentration (CMC), which can be defined as the lowest concentration atwhich the dissolved macromolecules begin to self-assemble. By selectionof the hydrophilic and other domains, drug delivery can be controlledand enhanced.

In one embodiment, the macromers are provided with at least onehydrophobic zone, and can form micelles including a region in whichhydrophobic materials will tend to bind and thus tend to reduce escapeof the drug from the formed gel. The hydrophobic zone may be enhanced byaddition of materials, including polymers, which do not contribute tothe formation of a gel network but which segregate into such zones toenhance their properties, such as a fatty acid, hydrocarbon, lipid, or asterol.

The ability of the macromonomers in one embodiment to form micellarhydrophobic centers not only allows the controlled dissolution ofhydrophobic bioactive compounds but also permits the hydrogel toselectively “expand” and “contract” around a transition temperature.This provides an “on-off” thermocontrol switch which permits thethermally sensitive delivery of drugs.

The cell membrane is composed of a bilayer with the inner region beinghydrophobic. This bilayer is believed to have a fluid and dynamicstructure, i.e., hydrophobic molecules can move around in thisstructure. A hydrophobic tail incorporated in a macromer can diffuseinto this lipid bilayer and result in the rest of the macromonomer(thus, the hydrogel) to better adhere to the tissue surface (see FIG.11). The choice of molecular group to be used as hydrophobic tail isguided by the fatty acid composition of the bilayer to assure minimumperturbation of the bilayer structure. Examples of suitable groups arefatty acids, diacylglycerols, molecules from membranes such asphosphatidylserine, and polycyclic hydrocarbons and derivatives, such ascholesterol, cholic acid, steroids and the like. Preferred hydrophobicgroups for this purpose are normal constituents of the human body. Thesemolecules will be used at a low concentration relative to nativemolecules in the membrane.

Use of macromers carrying one or more hydrophobic groups can improve theadherence of a hydrogel to a biological material by anchoring a segmentof the hydrogel in the lipid bilayer. This anchoring will cause minimalperturbation to the underlying tissue because the insertion of the fattyacid terminal of the macromer into the lipid membrane involves purelyphysical interaction. The macromer may be applied by using a prewash ofthe surface with these molecules, in effect ‘preparing’ the surface forcoupling and/or an in situ photopolymerization of a mixture of theselipid-penetrating molecules with the crosslinkable macromers.

The hydrophobic region may include oligomers of hydroxy acids such aslactic acid or glycolic acid, or oligomers of caprolactone, amino acids,anhydrides, orthoesters, phosphazenes, phosphates, polyhydroxy acids orcopolymers of these subunits. Additionally the hydrophobic region may beformed of poly(propylene oxide), poly(butylene oxide), or a hydrophobicnon-block mixed poly(alkylene oxide) or copolymers thereof.Biodegradable hydrophobic polyanhydrides are disclosed in, for example,U.S. Pat. Nos. 4,757,128, 4,857,311, 4,888,176, and 4,789,724, thedisclosure of which is incorporated by reference herein. Poly L-lactide,or poly D,L-lactide for example may be used. In another embodiment thehydrophobic region may be a polyester which is a copolymer ofpoly(lactic-co-glycolic)acid (PLGA).

The macromer also may be provided as a mixture including a hydrophobicmaterial non-covalently associated with the macromer, wherein thehydrophobic material is, for example, a hydrocarbon, a lipid, a fattyacid, or a sterol.

Hydrophilic Regions.

Water soluble hydrophilic oligomers available in the art may beincorporated into the biodegradable macromers. The hydrophilic regioncan be for example, polymer blocks of poly(ethylene glycol),poly(ethylene oxide), poly(vinyl alcohol), poly(vinylpyrrolidone),poly(ethyloxazoline), or polysaccharides or carbohydrates such ashyaluronic acid, dextran, heparan sulfate, chondroitin sulfate, heparin,or alginate, or proteins such as gelatin, collagen, albumin, ovalbumin,or polyamino acids.

Biodegradable Regions

Biodegradable molecules or polymers thereof available in the art may beincorporated into the macromers. The biodegradable region is preferablyhydrolyzable under in vivo conditions. In some embodiments, thedifferent properties, such as biodegradability and hydrophobicity orhydrophilicity, may be present within the same region of the macromer.

Useful hydrolyzable groups include polymers and oligomers of glycolide,lactide, epsilon-caprolactone, other hydroxy acids, and otherbiologically degradable polymers that yield materials that are non-toxicor present as normal metabolites in the body. Preferredpoly(alpha-hydroxy acids) are poly(glycolic acid), poly(DL-lactic acid)and poly(L-lactic acid). Other useful materials include poly(aminoacids), polycarbonates, poly(anhydrides), poly(orthoesters),poly(phosphazines) and poly(phosphoesters). Polylactones such aspoly(epsilon-caprolactone), poly(delta-caprolactone),poly(delta-valerolactone) and poly(gamma-butyrolactone), for example,are also usefull. The biodegradable regions may have a degree ofpolymerization ranging from one up to values that would yield a productthat was not substantially water soluble. Thus, monomeric, dimeric,trimeric, oligomeric, and polymeric regions may be used.

Biodegradable regions can be constructed from polymers or monomers usinglinkages susceptible to biodegradation, such as ester, peptide,anhydride, orthoester, phosphazine and phosphoester bonds. The timerequired for a polymer to degrade can be tailored by selectingappropriate monomers. Differences in crystallinity also alterdegradation rates. For relatively hydrophobic polymers, actual mass lossonly begins when the oligomeric fragments are small enough to be watersoluble. Thus, initial polymer molecular weight influences. thedegradation rate.

Therapeutic Applications

Biodegradable, temperature responsive hydrogels can be formed in situand may be use in a variety of therapeutic applications includingsurgical applications. In one embodiment the gels can be appliedtopically to the skin to treat a variety of conditions such as abrasion,keratoses, inflammatory dermatoses, injury resulting from a surgicalprocedure, and disturbed keratinization. The hydrogels may includetherapeutic agents such as antibiotics, or antifungals for the localizedtreatment of different skin conditions.

Macromers which are liquid at room temperature and gel at bodytemperature, such as macromers including a Pluronic™ poloxamer, may beused in treatment of burns and other external injuries. The hydrogelsare useful in burn applications, since the hydrogel layer formed on theskin provides local or transdermal delivery of drug to the burn site;maintains high moisture levels on severely burned sites, thusdiminishing dehydration; adheres strongly to the damaged tissue, and iselastic, thus minimizing delamination and “peeling” of the hydrogeldressing; and absorbs exudate from the wound. Hydrogels may be selectedwhich dissolve into components which are absorbable and non-toxic, whichpromote healing, and gel spontaneously and quickly on the burn site,prior to optional further crosslinking.

The macromers also may be applied to biological tissue, or on thesurface of a medical device, to form hydrogels in a variety of surgicalapplications for the treatment of tissue or organs. The gel also may beapplied between two surfaces, such as tissue surfaces, to adhere thesurfaces. The hydrogels may be applied to tissue such as vasculartissue, for example for the treatment of restenosis of the arteries orin angioplasty procedures A biologically active material may be providedin the gel optionally in the form of particles, microparticles, pro-drugconjugates, or liposomes. The macromers may be designed such that thecrosslinked gel changes in permeability in response to a change intemperature, ionic concentration or a change in pH, thereby altering therate of drug release from the hydrogel.

Drug Delivery

The macromers may be crosslinked reversibly or irreversibly to form gelsfor controlled drug delivery applications. The composition andproperties of the macromers can be selected and fabricated to producehydrogels with desired drug delivery properties. The drug may beprovided in the macromer solution prior to or after administration, andeither before or after gel formation, depending on the macromercomposition.

For example, the gels can be designed to have a selected rate of drugrelease, such as first order or zero order drug release kinetics. Forspecific drugs, such as peptides, the composition of the gel may bedesigned to result in pulsatile or mixed wave release characteristics inorder to obtain maximum drug efficacy and to minimize side effects andtolerance development. Bae et al., Pharmaceutical Research, 8: 531(1991).

The drug release profiles can be selected by the use of macromers andgels formed therefrom that respond to specific external stimuli such asultrasound, temperature, pH or electric current. For example, the extentof swelling and size of these hydrogels can be modulated. Changesinduced in the swelling directly correlate to the rate of release of theincorporated drugs. Through this, a particular release profile may beobtained. The hydrogels are preferably biodegradable so that removal isnot required after administration or implantation.

The gels permit controlled drug delivery and release of a biologicallyactive agent in a predictable and controlled manner locally at thetargeted site where it is needed, when the tissue to be treated islocalized. In other embodiments, the gels also can be used for systemicdelivery.

A variety of therapeutic agents can be delivered using the hydrogels.Examples include synthetic inorganic and organic compounds, proteins andpeptides, polysaceharides and other sugars, lipids, gangliosides, andnucleic acid sequences having therapeutic, prophylactic or diagnosticactivities. Nucleic acid sequences include genes, antisense moleculeswhich bind to complementary DNA to inhibit transcription, and ribozymes.The agents to be incorporated can have a variety of biologicalactivities, such as vasoactive agents, neuroactive agents, hormones,anticoagulants, immunomodulating agents, cytotoxic agents, antibiotics,antivirals, antisense, antigens, and antibodies. Proteins includingantibodies or antigens can also be delivered. Proteins are defined asconsisting of 100 amino acid residues or more; peptides are less than100 amino acid residues. Unless otherwise stated, the term proteinrefers to both proteins and peptides. Examples include insulin and otherhormones.

Specific materials include antibiotics, antivirals, antiinflammatories,both steroidal and non-steroidal, antineoplastics, anti-spasmodicsincluding channel blockers, modulators of cell-extracellular matrixinteractions including cell growth inhibitors and anti-adhesionmolecules, enzymes and enzyme inhibitors, anticoagulants and/orantithrombotic agents, growth factors, DNA, RNA, inhibitors of DNA, RNAor protein synthesis, compounds modulating cell migration, proliferationand/or growth, vasodilating agents, and other drugs commonly used forthe treatment of injury to tissue. Specific examples of these compoundsinclude angiotensin converting enzyme inhibitors, prostacyclin, heparin,salicylates, nitrates, calcium channel blocking drugs, streptokinase,urokinase, tissue plasminogen activator (TPA) and anisoylatedplasminogen activator (TPA) and anisoylated plasminogen-streptokinaseactivator complex (APSAC), colchicine and alkylating agents, andaptomers. Specific examples of modulators of cell interactions includeinterleukins, platelet derived growth factor, acidic and basicfibroblast growth factor (FGF), transformation growth factor β (TGF β),epidermal growth factor (EGF), insulin-like growth factor, andantibodies thereto. Specific examples of nucleic acids include genes andcDNAs encoding proteins, expression vectors, antisense and otheroligonucleotides such as ribozymes which can be used to regulate orprevent gene expression. Specific examples of other bioactive agentsinclude modified extracellular matrix components or their receptors, andlipid and cholesterol sequestrants.

Examples of proteins further include cytokines such as interferons andinterleukins, poetins, and colony-stimulating factors. Carbohydratesinclude Sialyl Lewis® which has been shown to bind to receptors forselectins to inhibit inflammation. A “Deliverable growth factorequivalent” (abbreviated DGFE), a growth factor for a cell or tissue,may be used, which is broadly construed as including growth factors,cytokines, interferons, interleukins, proteins, colony-stimulatingfactors, gibberellins, auxins, and vitamins; further including peptidefragments or other active fragments of the above; and further includingvectors, i.e., nucleic acid constructs capable of synthesizing suchfactors in the target cells, whether by transformation or transientexpression; and further including effectors which stimulate or depressthe synthesis of such factors in the tissue, including natural signalmolecules, antisense and triplex nucleic acids, and the like. ExemplaryDGFE's are vascular endothelial growth factor (VEGF), endothelial cellgrowth factor (ECGF), basic fibroblast growth factor (bFGF), bonemorphogenetic protein (BMP), and platelet derived growth factor (PDGF),and DNA's encoding for them. Exemplary clot dissolving agents are tissueplasminogen activator, streptokinase, urokinase and heparin.

Drugs having antioxidant activity (i.e., destroying or preventingformation of active oxygen) may be provided in the hydrogel, which areuseful, for example, in the prevention of adhesions. Examples includesuperoxide dismutase, or other protein drugs include catalases,peroxidases and general oxidases or oxidative enzymes such as cytochromeP450, glutathione peroxidase, and other native or denaturedhemoproteins.

Mammalian stress response proteins or heat shock proteins, such as heatshock protein 70 (hsp 70) and hsp 90, or those stimuli which act toinhibit or reduce stress response proteins or heat shock proteinexpression, for example, flavonoids, may be provided in the hydrogel.

The macromers may be provided in pharmaceutical acceptable carriersknown to those skilled in the art, such as saline or phosphate bufferedsaline. For example, suitable carriers for parenteral adminstration maybe used.

Administration of Macromers

Modern surgical procedures which provide access to a variety of organsusing minimally invasive surgical devices may be used to apply themacromers. Using techniques such as laparoscopy/endoscopy, it ispossible to deposit a macromonomer solution at a localized site andsubsequently polymerize it inside the body. This method of “on-site”polymerization offers unique advantages such as conformity to specificorgans and adherence to underlying tissue. Hill-West J. L. et al.,Obstetrics & Gynecology, 83:59 (1994). Catheter delivery systemsavailable in the art also may be used as described, for example, in U.S.Pat. Nos. 5,328,471 and 5,213,580 to Slepian. The macromer also mayapplied during surgery conducted through the cannula of a trocar.

Formation of Microspheres

In one embodiment, the biodegrabable macromers are crosslinked, eitherreversibly or nonreversibly to form microspheres. As used herein, theterm “Microspheres” includes includes particles having a uniformspherical shape or an irregular shape, and microcapsules (having a coreand an outer layer of polymer) which generally have a diameter from thenanometer range up to about 5 mm. In a preferred embodiment, themicrospheres are dispersed in biocompatible, biodegradable hydrogelmatrices. The microspheres are useful for controlled release andtargeted delivery of drugs within the body.

The microspheres are formed in one embodiment by aggregation andsubsequent polymerization of portions of the macromers which are similarin charge properties such as hydrophilicity. This results in a matrixwhich consists of spontaneously-assembled “nodes”, which may becrosslinked covalently, and may be further covalently linked tohydrophilic bridges of the macromers to form a hydrogel.

When the macromer is amphiphilic and includes hydrophobic andhydrophilic domains, in an aqueous environment, at or above a certainconcentration, the molecules to arrange themselves into organizedstructures called micelles, at the critical micellar concentration(CMC). These micelles can be of different shapes and sizes, though aregenerally spherical or elliptical shape. When the solution is water, thehydrophobic portions are at the center of the micelle while thehydrophilic tails orient themselves toward water. The interior core of atypical surfactant has a size from 10-30 Angstroms. Pluronic™ poloxamerbased biodegradable macromers, as described in Example 1, undergomicellization in an aqueous environment with CMC values ranging between0 and 5% (w/v). After photopolymerization and gelation, this micellarstructure is preserved in the crosslinked gel. On a microscopic level,the gel contains micelles which are interconnected by covalent bonds toform the gel. These micellar domains or microspheres can be used for thecontrolled or sustained release of drugs. A schematic representation ofsuch a material is shown in FIG. 12. Controlled, pseudo-zero orderrelease of small compounds such as chlorohexidine is possible from suchhydrogels.

The hydrogel thus is formed in one embodiment by providing a solution ofmacromer in aqueous solution (with or without drug); “freezing” themicellar structure of the macromer by a chemical crosslinking via achemical reaction; adding the drug to the crosslinked macromer if it hasnot been already added; and using the resultant dispersed composite,containing microspheres consisting of drug-attracting micellar cores,for drug delivery.

In addition to photopolymerization, crosslinking can be implemented by,for example, isocyanate-amine chemistry, or hydroxy- or aldehyde-aminechemistry, to freeze micellar structure. For example, isocyanateterminated poloxamer lactate diol can react in water to form crosslinkedpolyurethane based networks. This is an advantageous method of forming adrug delivery device for local or systemic delivery, because theformation of the delivery-controlling micropheres and themicrosphere-confining gel is accomplished simultaneously, and may beaccomplished at the site of delivery in a few seconds byphotopolymerization.

In one embodiment the macromer includes PEO segments, and hydrophobic“ends” containing reactive groups, and the micellar domains arehydrophobic and are interlinked by the PEG segments to form a hydrogel.Reversible gelling microsphere—forming macromers also may be made fromPluronics™ (PEG-PPO-PEG), lactylated and acrylate-capped, which aregelled and reacted in a non-aqueous phase. A hydrophilic drug then maybe added (while in the hydrophobic solvent) which partitions to thehydrophilic core. Because the micelles have been cross-linked in thehydrophobic environment, they will not be able to revert to theconformation which they would normally assume in a hydrophilicenvironment. The trapped hydrophilic drug molecules then need to diffusethrough a relatively hydrophobic region to escape from the nanoparticle.This permits flexibility in the formation of microspheres. They may behydrophilic or hydrophobic depending on the solvent in which they arepolymerized, and on the composition of the macromers.

In other embodiments, physical or chemical crosslinking to formhydrogels (or organogels) can occur in zones other than thoseresponsible for the primary sustained release characteristics of thematrix. For example, “single-ended” materials could have alternativereaction sites on the non-micellar ends, which could subsequentlyreacted to form a gel. Since matrix-controlled drug delivery is afunction of both diffusion from the micelles and of matrix degradation,manipulation of the macromolecular backbone can also control matrixdegradation. This can occur through stabilization of hydrolytic groupsby their chemical and physical environment (for example, macromers basedon reverse Pluronic™ gels are more stable than normal Pluronic™ gels, inaqueous solution). It is possible that the increased hydrophobicity ofthe environment of the lactide ester bonds, due to the adjacent blockbeing PPO rather than PEO, inhibits hydrolysis of the bond.

Alternatively, and particularly in gel-forming compositions, thecross-linking reactive groups or biodegradable groups may be in thehydrophilic portions of the macromers, so that the hydrophobic domainswould not be locally crosslinked in the hydrophobic regions, while themicelles would still be stabilized by the crosslinking of the material,and particular hydrophobic sections of macromers would be stericallyrestricted to one or only a few different micelles. In either of thesecases, the hydrophobic zones are not rigidly crosslinked, but areconnected to crosslinks via the hydrophilic blocks, which may be veryflexible. The hydrophobic blocks thus can associate above or below acritical temperature, and dissociate on change in temperature. Thisallows, for example, both thermosensitive gelation and thermosensitivevariation in drug diffusion rate.

The hydrogels may be designed to be biodegradable by incorporation of agroup such as a lactide, glycolide or other self-degrading linkage.Alternatively, this is not necessary when non-gelled nanospheres areformed, since these are small enough to be removed by phagocytosis.Control of the rates of delivery of both small and large molecules canbe obtained by control of the hydrophobicity of the associatinghydrophobic domains of amphipathic hydrogels.

The crosslinked microspheres containing a biologically active agent, ineither gel or dispersion form, can be made in a single step. In additionto drug delivery applications, the method is suitable for non-medicaluses including delivery of agricultural materials such as herbicides andpesticides and in water treatment.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1 Synthesis and Thermal Responsiveness ofF127-(Lactate)6-Acrylate

a) Synthesis.

F127-(lactate)0-acrylate (unlactated control) (=F127A2?) was synthesizedby acrylating 100 g of Pluronic™ F127 (polypropylene oxide-polyethyleneoxide block copolymer, BASF, mol. wt. 12000)(“F127”) in anhydroustoluene using triethylamine and acryloyl chloride, in an argonatmosphere at 60° C. for 10 minutes. The hot, turbid reaction mixturewas filtered and the filtrate was added to a large excess of hexane. Themonomer was collected by vacuum filtration and dried in vacuum to aconstant weight.

F127-(lactate)6-acrylate was synthesized as follows. F127 was melt driedin vacuo at 100° C. for 4 hours. D,L-lactide (Boehringer Ingelheim) wasadded to the melt under a nitrogen flush, followed by stannous octoateas a ring opening catalyst. After a reaction time of 4 hours, the meltwas dissolved in toluene and precipitated in a large excess of hexane.Acrylation of F127-(lactate)6 was carried out as described above for theacrylation of F127-(lactate)0-acrylate. All macromonomers werecharacterized by NMR and HPLC.

The relationship between the macromer, the thermally-reversible(physical) gel, and the irreversible (crosslinked) gel is shown in FIG.1.

b) Measurement of the Sol-Gel Transition as a Function of Concentrationand Temperature.

Thermoreversible gel formation of the aqueous solutions of themacromonomers at a certain transition temperature was demonstrated. Thistransition temperature was recorded as a function of temperature andconcentration. The results demonstrated that sol-gel transition can becontrolled through the incorporation of hydrophobic lactyl units.

Transition temperature as a function of concentration was determined bypreparing 20% w/v aqueous solutions of F127-(lactate)0-acrylate andF127-(lactate)6-acrylate as stock solutions. 15% (w/v), 12.5% (w/v), 10%(w/v) and 5% (w/v) macromonomer aqueous? solutions in screw cap vialswere prepared by dilutions of the stock solutions. The solutions wereallowed to equilibrate at 25° C. The vials were inverted and observedfor fluid flow. The concentration at which no fluid flow was observedwas recorded (see Table 1).

The transition temperature as a function of temperature was determinedby preparing 10% (w/v) aqueous solutions of F127-(lactate)6-acrylate andF127-(lactate)0-acrylate and equilibrating them at room temperature.(The concentration of the solutions are wt/vol % in aqueous solutionunless otherwise stated.) The sample vials were immersed in atemperature controlled bath and the fluid flow was observed at differenttemperatures. The temperature at which no fluid flow was observed wasrecorded (see Table 1). TABLE 1 Sol-Gel Sol-Gel Transition (% TransitionMacromonomers w/v)** (° C.)*** F127-(Lactate)0- 30 40 AcrylateF127-(Lactate)6- 10 25 Acrylate**Sol-Gel Transition as a function of concentration (temperature 25°C.).***Sol-Gel Transition of 10% w/v solutions as a function of temperature.

c) Polymerization and Measurement of Hydrogel Dimensions.

A 10% solution of F127-(lactate)6-acrylate in PBS (phosphate bufferedsaline) was polymerized using long wave UV light. The polymerizationswere performed in a cylindrical plastic mold Darocur™ 2959 (Ciba Geigy)was used as the photoinitiator. The hydrogel was allowed to reachequilibrium swelling by immersing in PBS for 24 hours at ambienttemperature. The change in dimension of the hydrogel at temperaturesranging from 0-50° C. was measured using vernier calipers, and is shownin FIG. 2. At low temperatures, the hydrophobic PPO (polypropyleneoxide) segments of the hydrogel may dissolve and swell, and increase thedimensions of the gel. At high temperatures, the PPO segments may becomehydrophobic and collapse into micromicellar hydrophobic domains, whichexclude water resulting in reduced swelling and smaller dimensions.

d) Degradation Experiments.

Hydrogels were prepared using 10% macromonomer solution as mentionedbefore and the degradation of hydrogel was monitored gravimetrically atvarious intervals of time. The experiments were performed at 37° C. inPBS. The lactate based photopolymerized hydrogel completely degraded in22 days (at 37° C., in PBS).

Thus, the macromers can be photopolymerized to form thermoresponsivehydrogels which degrade under physiological conditions.

The macromers and related prior art materials are referred to herein inthe form XXXLLAA, where XXX is either part of the trade name of aprecursor polymer (e.g., L81 for Pluronic™ L81 poloxamer) or refers toanother property of the base polymer (e.g., 8K for 8,000 nominal DaltonPEO). LL denotes the terminal block, typically of a degradable hydroxyacid (e.g., L5 denotes an average of 5 lactate residues per arm of thepolymer), where L, G, C and TMC or T represent, respectively, lactate,glycolate, epsilon-caproate, and trimethylenecarbonate. AA represents aterminal group; for example, A is for acrylate, so A2 would represent 2acrylate terminations on the macromer as a whole.

EXAMPLE 2 Dextran Release by F127A2

The non-degradable material, F127A2, was made as described above inExample 1, with no addition of hydroxy acid to the Pluronic™ polymerbackbone. Dextran (labeled with fluorescein) of molecular weight 71,000daltons was mixed at 1% final concentration with F127A2 macromer (finalconcentration 10% wt/vol, in water) and polymerized as described inExample 1. Release of dextran was determined by visible absorbance.Release kinetics were significantly altered by temperature, as shown inFIG. 3.

EXAMPLE 3 Synthesis of Macromers with Biodegradable Linking Groups

Four monomer types were made by the general procedures described inExample 1, each containing about 4 units of each of four differentbiodegradable linkers, designated by L (lactate), C (caprolactone), G(glycolide), and TMC (trimethylene carbonate). Parameters for thesynthesis of the thermosensitive macromonomers are listed in Table 2.Properties of the monomers characterized are listed in Table 3,including biodegradable segment and end group incorporation by HPLC andNMR, and Mn determined by GPC and NMR. TABLE 2 Temp Feed Ratio ° C./M.W. PPO PEO Monomer/ time Yield Compound (g/mole) M.W. M.W. diol (h)(g) F127L4A2 12600 3780 8820 4 180-190/5 80.46 F127C4A2 12600 3780 88204 180-190/5 81.38 F127G4A2 12600 3780 8820 4 180-190/5 71.89 F127TMC4A212600 3780 8820 4 180-190/5 79.29

TABLE 3 Biodeg. Biodeg. End End Seg. Seg. Group Group Mn Mn Mn Macro-Incorp. Incorp. Incorp. Incorp. GPC NMR Expected monomer (HPLC) (MNR)(HPLC) (NMR) g/mol g/mol g/mol F127L4A2 5.68 ± 0.01 5.58 2.09 ± 0.012.00 10800 11316 12998 F127G4A2 5.39 ± 0.02 5.04 2.05 ± 0.02 2.31 1080010804 12942 F127C4A2 5.49 ± 0.02 5.45 2.09 ± 0.03 2.11 10000 13062 13166F127TMC4A2 — 3.26 2.08 ± 0.03 2.09 12100 NA —

The monomers differed in their rate of polymerization and rate ofdegradation. The long UV photopolymerization profiles are shown in FIG.4. The in vitro degradation profiles of the crosslinked hydrogels areshown in FIG. 5.

The macromers had similar biocompatibility profiles, as shown in FIG. 6,as measured by the HFF cell adhesion test. In FIG. 7, release rates offluorescent dextran at 37° C. and 0° C. is shown for a prior artmaterial (F127A2) and for macromers with degradable hydrophobic blocksformed of lactide (F127L4A2), glycolide (F127G4A2) and caprolactone(F127C4A2). A longer period of quasi-zero order delivery, after theinitial burst, and a distinct difference in the rates of efflux betweenthe lower and higher temperatures, is obtained with the macromersincluding the degradable blocks, in comparison to the prior artmaterial. In FIG. 8, the transition temperatures (for volume change andchange of dextran release rate) are shown as a funtction of macromerconcenration in the gel for the above materials and also a trimethylenecarbonate based material (F127TMC4A2), a “reverse” meroxapol materialwith lactide (25R8L4A2), and a “normal” material (F68L4A2) of equivalenthydrophobicity.

The HFF test was conducted as follows:

a) Preparation of Gel.

0.5 gram of test material was dissolved in 4.5 ml standardreconstitution solution (Irgacure 1200 ppm, 3% Pluronic F127). Thesolution was filter sterilized using 0.2 micron filter. In a sterilehood, a glass coverslip (18 mm sq) was sterilized using 70% ethanol andwas placed in a 6 well, 35 mm tissue culture dish. 200 μL of the sterilemacromonomer solution was spread on a sterile coverslip. The solutionwas then exposed to long wavelength UV light (Black Ray, 20 mW/cm2, 1minute) to form a gel.

c) Preparation of Cell Suspension.

Human foreskin fibroblasts (HFF) cells were purchased from ATCC. Cellswere used at a passage 22-23. HFF cells were cultured in a standardtissue culture ware in a humidified atmosphere containing 5% CO₂. Cellswere detached from the culture flask using a 3 ml trypsin/EDTA solution(0.05% /0.53 mm) and centrifuged (2500 rpm, 3 minutes). The cell pilotwas resuspended in cell culture medium (DMEM+10% FCS) at a concentrationof 250000 cells/ml.

d) Cell Attachment Assay.

The gels were washed with 3 ml DMEM (Dulbecco's Modified Eagles' Medium)solution and then seeded with 25000 cells/cm2 cell density. After 18 h,the gel surface and tissue culture polystyrene surface were observedunder microscope and photographed. The gels were separated fromcoverslip and transferred into a new petri dish. The cells adhered tothe gels were detached using 3 ml trypsin/EDTA (0.05% /0.53 mm)solution. A Coulter counter was used to determine the cell density.

EXAMPLE 4 Effects of Linking Group Hydrophobicity on Small MoleculeDelivery

Micelle-forming biodegradable macromers were synthesized andcharacterized which included a a non-thermosensitive core. The macromersillustrated the effects of hydrophobicity on delivery capacity for smallhydrophobic molecules. The macromers were formed by synthesizingcopolymers of PEG (molecular weight 8000) with different combinations ofpolycaprolactone and polyglycolate which were then end capped withacrylate moieties. The structures are shown in FIG. 9, where p is thenumber of glycolic acid groups and q is the number of caprolactonegroups. Hydrophobicity of the mixed hydroxy acid blocks increases from Ato D. The ability of these monomers to solubilize model hydrophobicdrugs was demonstrated by a study of the CMC through the gradualdissolution of a molecular probe, 1,6 diphenyl 1,3,5-hexatriene (DPH).

Effect of Hydrophobicity on Drug Incorporation into Gels

a) Synthesis of Monomers.

The molecular structures of the monomers are shown in FIG. 9.Polyethylene glycol 8000 (Union Carbide) was melt-dried at 100-110° C.in vacuum (10-15 mm Hg) for 4-6 hours. Caprolactone (predistilled,Aldrich), and glycolide, were charged at appropriate ratios into aSchlenk-type reaction vessel and stannous 2-ethyl hexanoate (Sigma) wasadded as a ring opening catalyst. The reaction was carried out for 4hours in an inert atmosphere at 180° C. The reaction mixture was thencooled to 80° C., dissolved in toluene, precipitated in hexane and theproduct was collected by vacuum filtration. The product was redissolvedin toluene and dried by azeotropic distillation.

Acrylation was carried out by the dropwise addition of a 2 molar excessof acryloyl chloride and triethylamine under a nitrogen flush at 65° C.for 1 hour. By-product salts were removed by vacuum filtration. Theproduct was isolated by precipitation in a large excess of hexanefollowed by vacuum-filtration. The monomers were characterized by NMR ona Varian 300 MHz nuclear magnetic spectrometer.

b) Determination of Critical Micellar Concentrations.

The hydrophobic dye 1,6, diphenyl 1,3,5-hexatriene (Aldrich), (DPH),which demonstrates enhanced absorbance (356 nm) at the CMC due toassociative interactions, was used in this study. Alexandridis et al.,Macromolecules, 27:2414 (1994). A stock solution of DPH was prepared inmethanol (0.4 mM). Aqueous monomer solutions were prepared bydissolution in PBS and dilution to the desired concentrations. 10 μl ofthe dye solution were added to each vial with equilibration for at least1 hour. The absorption spectra of the polymer/dye/water solutions wererecorded in the 250-500 nm range using a Hitachi UV-VIS Spectrometer.

c) Photopolymerization.

Photopolymerization of the polymer solutions were carried out in bothvisible and ultraviolet light systems as described in: Sawhney A. S. etal., Macromolecules, 26: 581 (1993); and PCT WO 93/17669 by Hubbell J.A. et al.

d) In vitro Degradation.

200 μl of 10% monomer solution were UV polymerized to form a gel. Thedegradation of the hydrogels was monitored at 37° C. in PBS.

e) Results

In the synthesis, hydrophobic segments of the monomers were changed byusing various combinations of caproate and glycolate linkages in themolecule. The critical micellization point was obtained from the firstinflection of the absorption vs. concentration curve. The curves areshown in FIG. 10. It is evident from the curves that the solubility ofthe dye is enhanced with increasing concentration of the monomer. TheCMC values during aggregation and photopolymerization for variousmonomers are listed in Table 4. TABLE 4 Critical Gel* Time Gel** TimeMicellar Initiated Initiated Using Total Concentration Using UV VisibleLight Degradation Monomer (%) Light (secs) (secs) time (days) A 0.92 5.5± 0.4 8.9 ± 0.1 10 B 0.55 5.8 ± 0.1 8.2 ± 0.5 14 C 0.32 5.2 ± 0.2 9.8 ±0.4 16 D 0.28 4.6 ± 0.1 10.4 ± 0.3  44*2,2-Dimethoxy-2-phenylacetophenone as UV initiator, Long UV light, 20%monomer conc.**Eosin, triethanolamine initiating system; green light source, 20%monomer conc.

The CMC value is lowered with increase in caproate content of themonomer. This may be due to the tighter aggregation of the hydrophobiccaproate moieties. The fast gelling ability of these monomers under UVand visible light is illustrated in Table 4. The gel times range between4-12 seconds. The photopolymerized hydrogels degrade under aqueousconditions. The degradation times, i.e., times to substantially completedissolution, varied from 10-44 days, increasing with cap/gly ratio. Thefast gelation times of these monomers, their ability to dissolvehydrophobic solutes and their controlled degradation rates render themexcellent candidates for localized drug delivery.

EXAMPLE 5 Synthesis of Macromers Forming Liquid Crystal Phases

a) Synthesis of Macromers.

P105L4A2, P84L5A2 and T904L5A2 macromers were synthesized by standardprocedures, generally as described in Example 1, from commercial basepolymers (P105 Pluronic™ poloxamer; T904 Tetronic four-armed ionic-groupcontaining polaxamer; P84 Pluronic™ reverse poloxamer, or meroxapol).

b) Characterization of Optical Effects and Drug Release Properties.

Aqueous solutions were prepared, and observed for anomalous opticaleffects (“Schlieren”) without crosslinking. Rates of release of a drugwere observed, wherein the drug had a molecular weight about 500 D, andsubstantial water solubility, as well as a hydrophobic region.

Aqueous solutions of all three macromers formed “Schlieren” type liquidcrystalline phases at concentrations of 55% and higher, at roomtemperatures. A temperature study of the LC phases showed that the LCphases for P84L4A2 and T904L4A2 are not stable at temperatures higherthan 30-35° C. The LC phase for these two polymers “phase separates”into two phases at T>35° C., one being an isotropic polymeric phase thatis not transparent to light and another phase that seemed to consist ofwater. In contrast, a concentrated solution of P105L4A2 (75% w/v)displays a highly anisotropic LC phase that maintains its stability totemperatures up to 110° C.

Aqueous solutions of P105L4A2 (in high concentrations) formed a highlyanisotropic liquid crystalline phase (LC phase) that results in gooddrug entrapment to slow down release. It was also observed that P84L5A2and T904L5A2 had significant differences in the self-assemblingcharacteristics (LC). It is possible that the drug is entrapped in thestable, highly oriented LC Phase of a p105L4A2/water system. P84L4A2 andT904L4A2 form LC phases with water, but these phases are not stableabove 30-35° C. At higher temperatures, the drug as well as some of thewater are excluded from the polymeric domains.

EXAMPLE 6 Treatment of Burns

The pluronic poloxamer based macromonomers, such as F127-TMC acrylate,have a “paste-like” consistency at temperatures above 37° C., and haveflow characteristics at low temperatures A “cool” formulated solution,optionally containing an appropriate drug (such as an antibiotic) ispoured on a burn site, providing instant relief. At body temperatures,the formulation gels to a paste like consistency. The gel is thencrosslinked, preferably by the action of light on an includedphotoinitiator. The characterization of photopolymerized hydrogels ascarriers for therapeutic materials to influence wound healing isdescribed in Sawhney et al., “The 21st Annual Meeting of the Society forBiomaterials,” Mar. 18-22, 1995, San Francisco, Calif., Abstract, thedisclosure of which is incorporated herein by reference.

The hydrogel layer on the skin provides transdermal delivery of drug tothe burn site; maintains high moisture levels on severely burned sites,thus preventing dehydration; adheres strongly to the damaged tissue, andis elastic, thus preventing delamination and “peeling” of the hydrogeldressing; and absorbs exudate from the wound. After a suitable time,controlled by the nature of the lining group (trimethylene carbonate inthis example, giving a residence time of over a week), the gel willdissolve into components which are absorbable or innocuous. It has beendemonstrated in other experiments that related gel formulations, basedon a polyethyleneglycol backbone such as the material 8KL5A2 (i.e,. PEOof molecular weight 8,000, with 5 lactate groups on each end terminatedwith acrylate groups), do not retard the healing of full thicknessbiopsy wounds in rat skin. The pentablock polymer F127-TMC acrylate ofExample 3 is improved in comparison to the prior-art 8KL5A2 polyethyleneglycol-based triblock formula in that it gels spontaneously on the burnsite, and thus does not tend to run off the site before it can bephotocrosslinked.

EXAMPLE 7 Use of Hydrophobic Macromers to Increase Tissue Adherence

Use of macromers carrying one or more hydrophobic groups can improve theadherence of a hydrogel to a biological material. A macromer havinghaving this property was synthesised. The base polymer was a Tetronic™4-armed polymer based on ethylene diamine, where each arm is aPEG-PPO-PEG triblock copolymer. The base polymer was extended withlactide as previously described in Example 1, and then capped with abouttwo moles of palmitoyl chloride per mole of polymer, in order to capabout half of the arms. The remainder of the hydroxyls were capped withacroyl chloride, as described in Example 1. The resulting macromer wasdispersed in water and was polymerized in contact with tissue, to whichit adhered tenaciously.

EXAMPLE 8 Formation of Microspheres

Pluronic™ based biodegradable macromers made as described above above,such as the materials of Example 3, in an aqueous solution formedmicelles with a CMC value ranging from about 1% to 5% w/v. Afterphotopolymerization, the structure of the micelle is substantiallypreserved.

EXAMPLE 9 Synthesis of F127-Dimer Isocyanate-F127 Lactate Acrylate

Two molecules of a macromer diol (Pluronic F127) are coupled with onemolecule of a diisocyanate (dimer isocyanate) to produce higher di- andtri-functional alcohols, to provide macromers with high elasticity, highdistensibility and high tissue adherence.

The following reagents are used: Pluronic F127 (BASF lot # WPM N 581B,Mn=12200); dimer isocyanate (DDI-1410, Henkel Lot# HL 20037, %NCO=14.1%); and dibutyltin dilaurate.

Synthesis of F127-DDI-F127: 366 g of Pluronic F127 was heated to 100° C.under vacuum for four hours to produce a melt. DDI-1410 (8.94 g) anddibutyltin dilaurate (0.115 g) was added to the melt (melt temperature70° C.) and stirred vigorously for 4 hours. The mixture readilycrystallized when cooled. Product was a white waxy crystalline material.Theoretical molecular weight=24,996 Daltons.

Synthesis of F127-DDI-F127 Lactate₅ diol: 100 g of F127-DDI-F127 wasdried for 4 hours under vacuum at 100° C. 4.67 g of (D,L) Lactide wascharged to the reaction pot under an argon flush. Stannous 2-ethylhexanoate (0.5 mole percent) was added to the reaction. The melt wasvigorously stirred at 150° C. under argon for 4 hours. The product wasisolated by precipitation in hexane, followed by filtration. The productwas a white, crystalline, flaky material.

Synthesis of F127-DDI-F127 Lactates acrylate: 100 g of F127-DDI-F127Lactate₅ diol was charged into a 1000 ml three-necked reaction vessel.800 ml of toluene (Aldrich, 0.005% water content) was added to theflask. 50-75 ml of toluene was azeotroped off to ensure moisture freereactants. 2.427 ml of predistilled triethylamine, followed by 2.165 mlsof acryloyl chloride was added to the reaction mixture at 65° C. Afterone hour of reaction time, the turbid reaction mixture was filtered, andisolated into a white powder by precipitation into a large excess ofhexane. The product was collected by vacuum filtration and dried to aconstant weight.

Molecular structure determination was carried out by NMR, IR. Theproduct was found to be soluble in water and crosslinkable by visibleand UV light. Percent water uptake of fully cured 10% w/whydrogels=22.1%. Hydrogels formed by photopolymerization at 10%concentration while on dead bovine tissue were determined to begenerally well adherent.

P105-DDI-P105 lactate acrylate and L81-DDI-L81 lactate acrylate wassynthesized from the respective Pluronic poloxamer starting materials(P105,L81) by the procedure described above. These macromers wereinsoluble in water. They were used to encapsulate bioactive molecules inhydrophobic matrices to achieve sustained drug release.

EXAMPLE 10 Synthesis of F127-DDI-F127 Isophorone Isocyanate

The synthesis and polymerization of a macromer which crosslinks withoutinvolving free radical polymerization is demonstrated 50 g ofF127-DDI-F127 diol, prepared as in Example 9, was dissolved in 100 ml oftoluene in a three necked reaction flask. 90 ml of toluene was distilledoff azeotropically at 110° C. under argon. The flask was maintained at100° C. for 12 hours under vacuum (12 mm Hg). The reaction flask wasthen cooled to room temp, and 200 ml of dry methylene chloride was addedto the reaction flask. 0.445 g of isophorone isocyanate (Aldrich) wasadded (in a bolus) to the reaction flask at approximately 30° C. 0.15 gof dibutyltin laurate was added to the reaction mixture. The reactionmixture was stirred under argon at 30° C. for 12 hours, and precipitatedin 1000 ml of hexane (EM Sciences). White flakes were collected byvacuum filtration, and rinsed with 150 ml of hexane. The product wasdried in a vacuum oven to a constant weight. Characterization by NMR, IRshowed synthesis of the expected material.

The polymerizability of F127-DDI-F127 isophorone isocyanate wasevaluated. Partially dried product (0.16 g) was added to 1.44 g ofdeionized water. The product initially formed bubbles in contact withwater, then dissolved over approximately 3 days to form a viscoussolution. To test polymerizability, 200 mg of F127-DDI-F127 isophoroneisocyanate solution of polyethyleneimine in methylene chloride. Thesolution was stirred vigorously for a few seconds. A gelatineous productwas observed. Gel time: 5.9 seconds. Polyethyleneimine is believed tohave hemostatic properties; this formulation thus is potentiallysuitable for a topical wound dressing. In addition, structures formed ofthese materials can be used as drug depots.

EXAMPLE 11 Effect of Hydrophobicity on Drug Release Kinetics for BulkDevices

Macromers were synthesized having a wide range of hydrophobicitiesranging from 0-90% PPO content. All macromers were tested at 15%macromer concentration except those whose PPO content was greater than60% which were used neat. FIG. 13 shows the rate of release of a smalldrug from gels of these macromers. At 10 and 15% macromer loading(8KL10, prior art; 25R8L4A2, based on a “reverse” Pluronic polymer) andPPO content of less than 60% hydrophobic partitioning did not show asignificant effect on prolonging 500 Da sparingly soluble drug release.Devices prepared with neat macromers (PPO content>60%; P84L5A2 andL81L5A2, synthesized by general procedures as described above) showed asignificant ability of these highly hydrophobic, dense macromers toretard water permeation and drug dissolution. In the extreme case(L81L5A2; PPO content 90%), the release kinetics showed first orderrelease with half of the drug being released from the device over 17days with the remainder being eluted from the device over a total of 66days.

EXAMPLE 12 Effect of Polymer Hydrophobicity on Drug Diffusivity

Membranes of constant thickness were prepared from neat macromers ofExample 11, and used as the diffusion barrier in a two-compartmentdialysis cell. FIGS. 14 and 15 show the increase in the concentration of500 Da drug in the receptor side of the cell over time. The diffusioncoefficient calculation was based on the following relationship:D=J/(A*(ΔC/AΔχ)where D is the diffusion coefficient, J is the measured flux, A is theexposed area of the film, ΔC is the concentration gradient across thefilm and Δχ is the average film thickness. The diffusion coefficientsfor macromers having 50% (P105L5A2) or 90% (L81L5A2) relativehydrophobic domain and were calculated to 1.6×10⁻⁹ cm²/sec and5.63×10⁻¹⁰ cm²/sec, respectively. Thus, diffusion was faster in the morehydrophobic material, as expected for a drug of low water solubility.

EXAMPLE 13 Release of Tetracycline and Taxol

A 30% w/w solution of F127 trimethylene carbonate acrylate (as describedin Example 3) in phosphate buffered saline, pH7.4 was prepared. 3000 ppmDarocur® (Ciba Geigy) was incorporated in the solutions asphotoinitiator. Tetracycline (free base, crystalline, F.W. 444.44) wasincorporated in the macromer solution by equilibration for 12 hours at37 degrees C. Then, 200 microliters of the solution was crosslinked byUV light (10 W/cm2, full cure). In vitro release of tetracycline fromthe 200 microliter cured gel, after a brief rinse, was carried out in 5mls PBS, pH 7.4, 37° C. The PBS was exchanged daily to ensure “sink”conditions. The release profile is seen FIG. 16. After an initial burst,tetracycline was released steadily for nearly a week.

Taxol was incorporated into gels by similar procedures, except thatTween™ surfactant was used to solubilize the Taxol concentrate. Asimilar release pattern to that seen with tetracycline was observed.

EXAMPLE 14 Urethane-Containing Macromers

PEO of molecular weight 1450 was reacted with approximately 1 mole oflactide per end, using procedures described above, to give 1.4KL2. The1.4KL2 was weighed into a 100 ml flask (8.65 g) and 270 ml of driedtoluene was added. About 50 ml of toluene was distilled off to removeresidual water as the azeotrope, and the solution was cooled. Then 0.858g (825 microliter) of commercial 1,6 hexane-diisocyanate was added, andalso 1 drop of dibutyltindilaurate (ca. 0.02 g). The solution was at 60degrees at addition, and warmed to 70 degrees over about 10 minutes.Heat was applied to maintain the solution at about 75 degrees for about3.5 hours. NMR and IR spectra confirmed consumption of the diisocyanate,and the resulting solution was therefore expected to contain alternatingPEO and hexane blocks, linked by urethane linkages, and terminated byhydroxyls. This material can be capped with reactive end groups,optionally after further extension with hydroxy acids, to form areactive macromer. The urethane links and hexane blocks are present topromote tissue adherence.

Modifications and variations of the present invention will be obvious tothose skilled in the art from the foregoing detailed description. Suchmodifications and variations are intended to come within the scope ofthe following claims.

1. A pharmaceutical formulation of a gel-forming macromer, comprising amacromer comprising at least four covalently linked polymeric blocks, atleast one thermally sensitive region, and at least one covalentlycrosslinkable group, and wherein at least two of the blocks arehydrophobic, and at least two of the blocks are hydrophilic, saidmacromer being dispersed or solubilized in a pharmaceutically acceptableaqueous solution or suspension, whereby the formulation is capable ofreversibly transitioning from a liquid state to a gel state by anincrease in temperature.
 2. The formulation of claim 1 wherein thetransitioning occurs at a temperature in the range of 0° C. to 60° C. 3.The formulation of claim 1 wherein at least one hydrophobic blockaggregates in aqueous solution to form a micellar hydrophobic domain. 4.The formulation of claim 1 wherein the macromer further comprisesbiodegradable blocks.
 5. The formulation of claim 1 further comprising abiologically active material.
 6. The formulation of claim 5 wherein thebiologically active material is in the form of microparticles.
 7. Theformulation of claim 6 wherein the biologically active material is asynthetic inorganic compound, an organic compound, a protein, a peptide,a polysaccharide, a lipid, a ganglioside, or a nucleic acid.
 8. Theformulation of claim 1 wherein the macromer is present in theformulation at a concentration of at least about 2% by weight.
 9. Theformulation of claim 1 wherein the carrier is suitable for parenteraladministration.
 10. The formulation of claim 1 wherein the hydrophobicblock is polypropylene oxide or polybutylene oxide.
 11. The formulationof claim 1 wherein the hydrophilic block is polyethylene oxide,polyvinylalcohol, polyvinylpyrrolidone, or polyethyloxazoline.
 12. Theformulation of claim 1 wherein the hydrophilic block is a residue of apolysaccharide.
 13. The formulation of claim 12 wherein thepolysaccharide is dextran, heparin, heparin sulfate, chondroitinsulfate, hyaluronic acid, or alginate.
 14. The formulation of claim 1wherein the hydrophilic block is a residue of gelatin, collagen,albumin, ovalbumin, or polyaminoacid.
 15. The formulation of claim 4wherein the biodegradable block is a single, oligomeric or polymericresidue of glycolic acid, lactic acid, caprolactone, butyrolactone,valerolactone, or carbonate.
 16. The formulation of claim 1 wherein thecovalently crosslinkable group is an unsaturated group.
 17. Theformulation of claim 16 wherein the unsaturated group is a vinyl group,acrylate group, methacrylate group, diacrylate group, or cinnamategroup.